Nutrient composition for the preparation of streptomycin



Patented May 20, 1952 UNITED STATES PATENT OFFICE NUTRIENT COMPOSITIONFOR THE PREPARATION OF STREPTOMYOIN No Drawing. Application December 30,1947, Serial No. 794,801

2 Claims. 1

This invention relates to the provision of a novel nutrient compositionuseful for culturing microorganisms for the production of antibioticsubstances, such as streptomycin, having particular reference to acomposition comprising vegetable oil meal, natural amino acids andinositol as essential ingredients.

A principal object of the invention is to provide a nutrient compositionwhich, when used in the culturing of microorganisms, will stimulate themicroorganisms to generate increased yields of substances which aredesired to be obtained.

Another object of the invention is the provision of a nutrientcomposition which, when used in the culturing of microorganisms,stimulates the microorganisms to form desirable fermentation products atan increased rate, whereby increased yields of such products per unittime are obtained.

An additional object of the invention is the provision of an improvednutrient composition which, when used in the culturing ofmicroorganisms, so influences the course of fermentation as to avoid theproduction of undesirable substances which are deleterious and trouble-,some in the subsequent isolation and purification processes.

A further object of the invention is the provision of a nutrientcomposition which, when used in the culturing of streptomycin-producingmicroorganisms, stimulates the microorganisms to generate increasedyields of streptomycin.

A still further object of the invention is the provision of an improvednutrient composition which, when used in the culturing ofstreptomycin-producing microorganisms, influences the course offermentation in such a manner as to avoid the production of undesirablehistaminelike bodies which are deleterious and troublesome in thesubsequent isolation and purification processes.

Other objects of the invention will in part be obvious and will in partappear hereinafter.

The invention accordingly comprises a composition of matter possessingthe characteristics, properties and the relation of components whichwill be exemplified in the composition hereinafter described and thescope of the application of which will be indicated in the claims.

In a biological process, as in any chemical process, increased emciencyin the transformation of raw material into the desired end product is ofprime importance. Where the yield in such process is small and the costof recovery 2 of the desired product from a large proportion of wastematerial is high, the yield becomes a controlling factor in the cost ofproduction.

In the production of antibiotic substances by microorganisms in presentfermentation processes, the yield of these substances is only a smallfraction of one percent of the weight of nutrient materials used in theculture media. Consequently, the desired substance in the spent culturemedium must be concentrated and isolated from relatively large volumesof liquor. This is commonly done by means of selective adsorption orslovent extraction processes. In addition to this, the fermentationprocesses are relatively slow, requiring several days to provide amaximum yield of product. As a result of these limiting factors, largeand numerous units of equipment are required to provide even smallquantities of purified product from a single fermentation. This is thesituation, for example, in the case of streptomycin production.

In the process for producing streptomycin, a suitable microorganism suchas Streptomyces grz'seus may be cultured in an aqueous medium containingrepresentative materials selected from three broad classes of thefollowing essential ingredients:

(a) An organic nitrogen source, such as peptone, beef extract, yeastextract, corn steepwater, vegetable protein material such as soybeanmeal, casein, enzyme-converted proteins, grain fermentation residues,and the like.

(b) A carbohydrate, such as glucose, corn syrup, starch, modifiedstarches. dextrins, soybean carbohydrate, lactose, maltose.

(c) A mineral supplement, such as sodium chloride, magnesium sulfate,ferrous sulfate, potassium phosphate, ammonium sulfate, potassiumsulfate, calcium carbonate, etc., or combinations of these.

The concentrations of substances from these classes in the medium may bevaried widely depending upon the combination chosen, but ordinarilyvaries between 0.5 percent and 2' percent, governed by the fermentationprocess being employed.

The culturing of microorganisms for the production of streptomycineither may be carried out by the surface-growth method or by thesubmerged-growth method. Both of these means have been employed inexperimental work but in a commercial process the organism is usuallygrown by the submerged method in large aerated tanks.

The yield of streptomycin is affected by many factors. Foremost of theseis the nutrient medium composition itself, which can vary in the nature,concentration and proportions of the individual components employed. Thebalance between the nitrogen, carbohydrate and mineral ingredients isvery sensitive and a combination of protein and sugars which give goodresults with one mineral supplement might be almost worthless with adifferent mineral supplement or a different proportion of the same one.This situation is complicated by the presence in most naturallyoccurring proteins and carbohydrates of various minera1 ingredients.Another factor influencing yield is the process of fermentation to bepracticed, that is to say, the selection of the proper physicalconditions. A medium that gives optimum results under one set ofoperating conditions may be inferior in another. 7 agitation,temperature, time and type of equip ment are important variables.

Soybean meal, with suitable carbohydrate and mineral supplements, hasproven to be one of the most desirable organic nitrogen sources becauseit gives relatively high yields of streptomycin. These increased yields,based on the weight of nutrient materials used, are still so low,however, that the production cost of streptomycin is undesirably high.

By vegetable oil meal is meant the residues obtained after the oiltherein has been removed by such means as, for example, mechanicalpressure or solvent extraction. To provide my novel composition, 100parts of such meal may be mixed with 0.05 to 50' per cent (preferably, 1to 10 per cent) of amino acids, and 0.01 to 10 per cent (preferably, 1to per cent) of inositol.

Soybean meal, or soybean protein, is preferred in practicing the presentinvention because of its availability, low cost, and the fact that ityields no histamine-like bodies when used as a nutrient composition inthe culturing of Streptomyces g'r'z'seus in the production ofstreptomycin.

Any of the natural amino acids, or combina? tions thereof, are suitablefor the practice of this invention. The amino acids referred to arethose which may be found, for example, in protein hy-' drolyzates. Apreferred amino acid material is a mixture of mono-amino mono-carboxylicamino acids which may be conveniently crystallized from neutralized andconcentrated hydrolyzates of corn, wheat, or soybean protein. Such amixture of amino acids consists chiefly of leucine, isoleucine',methionine, valine, alanine, tyrosine and phenylalanine.

Inositol (hexahydroxycyclohexane) is obtained by hydrolysis of phyticacid or its derivatives, such as phytin. Numerous procedures foraccomplishing the hydrolysis, which involve heating the inositol esterwith aqueous acid or alkali, or with water alone, are described in thechemical literature. Particular reference is made to the article byBartow and Walker published in the Journal of Industrial and EngineeringChemistry,- volume 30, pages 300 to 303 (1938).

Since certain changes in carrying out the above process, and certainmodifications in the product which embody the invention may be madewith- Aeration,

out departing from its scope, it is intended that all matter containedin the above description shall be interpreted as illustrative and not ina limiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall therebetween.

Having described my invention What I claim as new and desire to secureby Letters Patent is:

1. A nutrient composition for the growth of streptomycin-producingmicroorganisms, which consists essentially of soybean meal and, asgrowth-promoting and streptomycin-production stimulating factors, freeinositol and a mixture of mono-amino, mono-carboxylic amino acidsobtained from a neutralizedand concentrated protein hydrolyzate selectedfrom the group consisting of corn, wheat and soybean proteinhydrolyzates, said inositol being present in the range of 0.01 to 10% byweight and said amino acids being present in a range of from 0.5 to 50by weight.

2. In the preparation of streptomycin by cul-' turingstreptomycin-producing microorganisms in an aqueous medium containingsoybean meal as the primary source of organic nitrogen, the improvementcomprising: adding to the medium, as growth-promoting andstreptomycin-production stimulating factors, free inositol and a mixtureof mono-amino, mono-carboxylic' amino acids obtained from a neutralizedand concentrated protein hydrolyz'ate selected from the group consistingof corn, wheat and soybean protein hydrolyzates, said inositol beingpresent in the range of 0.01 to 10% by weight and said amino acids beingpresent in a range of from 0.5 to 50% by weight based on the combinedweight of said soybean meal, inositol and amino acids.

WILLIAM F. ALLEN.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS OTHER REFERENCES Tatum: Annual Review ofBiochemistry, v01. XIII (1944) Stanford Univ. Press, pages 668, 669.

McMahon: Jour. Bact., #47, April 1944, pages

1. A NUTRIENT COMPOSITION FOR THE GROWTH OF STREPTOMYCIN-PRODUCINGMICROORGANISMS, WHICH CONSISTS ESSENTIALLY OF SOYBEAN MEAL AND, ASGROWTH-PROMOTING AND STREPTOMYCIN-PRODUCTION STIMULATING FACTORS, FREEINOSITOL AND A MIXTURE OF MONO-AMINO, MONO-CARBOXYLIC AMINO ACIDSOBTAINED FROM A NEUTRALIZED AND CONCENTRATED PROTEIN HYDROLYZATESELECTED FROM THE GROUP CONSISTING OF CORN, WHEAT AND SOYBEAN PROTEINHYDROLYZATES, SAID INOSITOL BEING PRESENT IN THE RANGE OF 0.01 TO 10% BYWEIGHT AND SAID AMINO ACIDS BEING PRESENT IN A RANGE OF FROM 0.5 TO 50%BY WEIGHT.